This invention relates generally to the field of growth factors, and more specifically to connective tissue growth factors (CTGF) and methods of modulating the activity of CTGFs.
Growth factors can be broadly defined as multifunctional, locally acting, intercellular signaling polypeptides which control both the ontogeny and maintenance of tissue form and function. The protein products of many of proto-oncogenes have been identified as growth factors and growth factor receptors. Normal versions of many oncogenes first discovered in mammals are also present in the genomes of organisms as disparate as yeast, drosophila, and frogs, and that they function during embryogenesis.
Growth factors stimulate target cells to proliferate, differentiate and organize in developing tissues. The action of growth factors is dependent on their binding to specific receptors which stimulates a signaling event within the cell. Examples of growth factors include platelet-derived growth factor (PDGF), insulin-like growth factor (IGF-I, IGF-II), transforming growth factor beta (TGF-xcex2), transforming growth factor alpha (TGF-xcex1), epidermal growth factor (EGF), acidic and basic fibroblast growth factors (AFGF, bFGF) and connective tissue growth factor (CTGF) which are known to stimulate cells to proliferate.
PDGF is a cationic, heat stable protein found in the alpha granules of circulating platelets and is known to be a mitogen and a chemotactic agent for connective tissue cells such as fibroblasts and smooth muscle cells. Because of the activities of this molecule, PDGF is believed to be a major factor involved in the normal healing of wounds and pathologically contributing to such conditions as atherosclerosis and fibrotic conditions. PDGF is a dimeric molecule consisting of combinations of xcex1 and/or xcex2 chains. The chains form heterodimers or homodimers and all combinations isolated to date are biologically active.
Studies on the role of various growth factors in tissue regeneration and repair have led to the discovery of PDGF-like proteins. These proteins share both immunological and biological activities with PDGF and can be blocked with antibodies specific to PDGF.
Polypeptide growth factors and cytokines are emerging as an important class of uterine proteins that may form growth signaling pathways between the maternal uterus and developing embryo or fetus. Studies in a variety of species have suggested that EGF, connective tissue EGF-like growth factor (HB-EGF), IGF-I, IGF-II, aFGF, bFGF, pleitrophin (PTN), leukemia inhibitory factor, colony-stimulating factor-1 (CSF-1), and TGF-xcex1 may be among the uterine growth-regulatory molecules involved in these processes.
CTGF is a cysteine-rich monomeric peptide of Mr 38,000, which is a growth factor having mitogenic and chemotactic activities for connective tissue cells. CTGF is secreted by cells and is active upon interaction with a specific cell-surface receptor. CTGF is the product of a gene unrelated to the xcex1 or xcex2 chain genes of PDGF. It is a member of a family of growth regulators which includes the mouse (also know as fisp-12 or xcex2IG-M2) and human CTGF, Cyr61 (mouse), Cef10 (chicken), and Nov (chicken). Based on sequence comparisons, it has been suggested that the members of this family all have a modular structure, consisting of (1) an insulin-like growth factor domain responsible for binding, (2) a von Willebrand factor domain responsible for complex formation, (3) a thrombospondin type I repeat, possibly responsible for binding matrix molecules, and (4) a C-terminal module found in matrix proteins, postulated to be responsible for receptor binding.
The sequence of the cDNA for human CTGF (HCTGF) contains an open reading frame of 1047 nucleotides with an initiation site at position 130 and a TGA termination site at position 1177 and encodes a peptide of 349 amino acids. There is only a 40% sequence homology between the CTGF cDNA and the cDNA for either the xcex1 or xcex2 chains of PDGF.
The hCTGF open reading frame encodes a polypeptide which contains 39 cysteine residues, indicating a protein with multiple intramolecular disulfide bonds. The amino terminus of the peptide contains a hydrophobic signal sequence indicative of a secreted protein and there are two N-linked glycosylation sites at asparagine residues 28 and 225 in the amino acid sequence. There is a 45% overall sequence homology between the CTGF polypeptide and the polypeptide encoded by the CEF-10 mRNA transcript; the homology reaches 52% when a putative alternative splicing region is deleted.
CTGF is antigenically related to PDGF although there is little if any peptide sequence homology. Anti-PDGF antibody has high affinity to the non-reduced forms of PDGF or CTGF, and ten-fold less affinity to the reduced forms of these peptides, which lack biological activity. This suggests that there are regions of shared tertiary structure between the PDGF isomers and the CTGF molecule, resulting in common antigenic epitopes.
The synthesis and secretion of CTGF are selectively induced by TGF-xcex2, BMP-2 and possibly other members of the TGFxcex2 superfamily of proteins. Although TGF-xcex2 can stimulate the growth of normal fibroblasts in soft agar, CTGF alone cannot induce this property in fibroblasts. However, it has been shown that the synthesis and action of CTGF are essential for the TGF-xcex2 to stimulate anchorage independent fibroblast growth.
It is probable that CTGF, or fragments thereof, functions as a growth factor in wound healing. Pathologically, CTGF has been postulated to be involved in conditions in which there is an overgrowth of connective tissue cells, such as systemic sclerosis, cancer, fibrotic conditions, and atherosclerosis.
The primary biological activities of CTGF polypeptide is its mitogenicity, or ability to stimulate target cells to proliferate and its role in the synthesis of the extracellular matrix. The ultimate result of this mitogenic activity in vivo, is the growth of targeted tissue. CTGF also possesses chemotactic activity, which is the chemically induced movement of cells as a result of interaction with particular molecules.
The present invention provides a polynucleotide and a polypeptide encoded thereby which has been identified as rat connective tissue growth factor (CTGF). In accordance with one aspect of the present invention, there is provided a novel recombinant CTGF, as well as active fragments, analogs and derivatives thereof.
In accordance with another aspect of the present invention, there are provided isolated nucleic acid molecules encoding the CTGF of the present invention including mRNA, DNA, cDNA, genomic DNA as well as active analogs and fragments of the protein.
In yet another aspect, the invention provides a method for producing a CTGF polypeptide by recombinant techniques comprising culturing recombinant prokaryotic and/or eukaryotic host cells, containing a nucleic acid sequence encoding a protein of the present invention, under conditions promoting expression of the protein and subsequent recovery of the protein. In a further aspect of the present invention, there are provided antibodies which bind to CTGFs.
In another aspect, the invention provides a polynucleotide for inhibiting expression of CTGF in a cell which comprises a contiguous nucleotide sequence complementary to a CTGF target nucleic acid sequence in a cell, and wherein the polynucleotide hybridizes to the CTGF target nucleic acid sequence thereby inhibiting expression of CTGF as compared to an uninhibited level of CTGF expression in the cell.
The invention further provides a method for inhibiting the expression of CTGF in a cell comprising contacting the cell with a polynucleotide containing a contiguous nucleotide sequence complementary to a CTGF target nucleic acid sequence in a cell, wherein the polynucleotide inhibits the expression of CTGF in the cell.
In accordance with yet a further aspect of the invention, there is provided a method for inhibiting the expression of CTGF in a subject comprising administering a polynucleotide containing a contiguous nucleotide sequence complementary to a CTGF target nucleic acid sequence in a cell to a subject, the polynucleotide is expressed at a level sufficient to inhibit expression of CTGF in the subject.
In another embodiment, the invention provides a pharmaceutical composition for the treatment of a disorder associated with CTGF. The pharmaceutical composition includes a pharmaceutically acceptable carrier and a therapeutically effective amount of an oligonucleotide that binds to a CTGF nucleic acid.